JPH0328190B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention provides a method for producing lipase by culturing lipase-producing microorganisms by feeding an organic nitrogen source mainly composed of amino acids or amino acids and peptides. A lipase suitable for catalyzing non-aqueous reactions such as oil and fat transesterification reactions, which is characterized by culturing while keeping the amino acid concentration in the liquid at a low concentration to increase the lipase production ability of lipase-producing microorganisms. Regarding the production method. [Prior Art] Conventionally, lipase, which is an enzyme that catalyzes the hydrolysis of fats and oils, has been known to use other hydrolytic enzymes such as Research on it is significantly behind compared to amylase, protease, etc. However, due to its unique substrate specificity, it also has many practical applications. for example
Rhizopusdelemar lipase is used in research in the fields of oleochemistry and biochemistry, such as determining the structure of fats and oils, due to its positional specificity for triglycerides, and is also used as a clinical diagnostic reagent because it acts well on blood lipids (lipoproteins). It is also expected to be applied as Furthermore, due to its fatty acid specificity, it is used in food processing and dairy flavor production. In addition to applying the hydrolysis reaction using lipase as described above, there are also industrial applications such as the synthesis of cosmetic fragrances using the reverse reaction, the synthesis reaction, and the high value-added conversion of oils and fats by transesterification of oils and fats. Its uses are wide. As described above, lipase, which is widely used as an enzyme, is widely distributed in nature, but from the viewpoint of enzyme resources, it cannot be obtained inexhaustibly from animal organs or plant seeds, and it cannot be obtained from water. Since it is difficult to extract and is unstable, industrial production relies on microbial sources as its source. [Means for Solving the Problem] In the process of investigating a method for culturing a microorganism that produces lipase that catalyzes the transesterification reaction of fats and oils, the present inventors added organic nitrogen to the medium for culturing the microorganism that produces lipase. It was also found that the productivity of lipase, that is, the transesterification ability of lipase-producing microorganisms, differs depending on the type of organic nitrogen source used. As a result of intensive research by the present inventors, we found that the reason for the change in lipase productivity depending on the type of organic nitrogen source used in the culture as described above is the amino acid content contained in the organic nitrogen source, that is, the culture It became clear that this was caused by the difference in amino acid concentration in the liquid. Microorganisms that produce lipase, such as Rhizopus, a heat-resistant strain of the genus Rhizopus.
In the culture of Rhizopus chinensis,
The assimilation rate of amino acids present in the medium changes according to the Michael-Menten equation. That is, among the amino acids present in the medium, amino acids with a high concentration have a high assimilation rate.
Among these assimilated amino acids, if the assimilation rate of amino acids other than leucine, phenylalanine, lysine, and arginine is high (the amino acid concentration in the medium is high), lipase will be produced by lipase-producing microorganisms. The present inventors have found that this is inhibited. Among these amino acids that inhibit lipase production, glutamic acid, proline, glycine, alanine, and aspartic acid have a significant inhibition on lipase production when their assimilation rate is high (the amino acid concentration in the medium is high). . On the other hand, with regard to amino acids such as leucine, phenylalanine, lysine, and arginine, no particular inhibitory effect is observed even if their assimilation rate is high (even if their concentration in the medium is high); On the contrary, it was found that even a promoting effect was observed. Therefore, when culturing a lipase-producing microorganism using an organic nitrogen source in a medium, the amino acid composition of the organic nitrogen source is important in order to increase the productivity of lipase by the lipase-producing microorganism.
In other words, among the amino acids in organic nitrogen sources, amino acids that inhibit lipase production, such as glutamic acid, proline, alanine, glycine, and aspartic acid, are either present in small amounts or do not exist as free amino acids even if their content is large. First, it is desirable that it be in the form of a peptide or the like. However, organic nitrogen sources commonly used for culturing microorganisms, such as yeast extract, meat extract, malt extract, and distillation waste concentrate (DDS), do not meet the above requirements, and organic nitrogen sources such as glutamic acid, alanine, glycine, and proline, etc. Contains a large amount of amino acids that inhibit lipase production, such as Therefore, even if the organic nitrogen sources mentioned above are used in general batch culture, it is not possible to increase the productivity of lipase by microorganisms. Therefore, to increase lipase productivity in batch cultures, natural protein sources, such as corn gluten meal, blood meal, feather meal or defatted soybean meal, are combined with proteases with appropriate substrate specificity, such as papain, pepsin or pancreatin. The organic nitrogen source must be produced by stopping the decomposition of amino acids that inhibit the production of lipase as described above in the form of peptides as much as possible. However, adding a new production process for an organic nitrogen source as described above to the culture process of lipase-producing microorganisms is economically disadvantageous and undesirable because it increases the production cost of lipase. The present invention is a method for increasing lipase productivity regardless of the type of organic nitrogen source used for culturing lipase-producing microorganisms.
Meat extracts, malt extracts, fish solvents, distillation waste concentrates (DDS), and the like can be used. Next, the present invention will be explained in more detail. [Effect] As mentioned above, in microorganisms that produce lipase, such as Rhizopus chinensis, a heat-resistant strain of the genus Rhizopus, the assimilation rate of amino acids follows the Michael-Menten equation, so for example, glutamic acid, proline, glycine, etc. By keeping the concentration of amino acids that inhibit lipase production, such as alanine and aspartate, in the medium low, the assimilation rate will be reduced.
The inhibitory effect is released. The present invention is a method for increasing the production of lipase by keeping the amino acid concentration in the medium low by feeding an amino acid source and culturing microorganisms while reducing the amino acid assimilation rate. We used naturally occurring organic nitrogen sources such as yeast extract, meat extract, malt extract, Proextract P (hydrolyzate of soybean meal and gluten), water-soluble and distillate waste concentrate (DDS). )
As a result of examining the relationship between various feeding amounts such as, the concentration of each amino acid in the medium, and lipase production, it was found that lipase activity increased only when culture was carried out at a feeding amount such that the total amino acid concentration in the medium was 5000 ppm or less. However, it was observed that the productivity of lipase increased rapidly. Looking at individual amino acids, amino acids that inhibit lipase production, other than leucine, phenylalanine, lysine, and arginine, inhibit lipase production when their individual concentrations exceed 1000 ppm in the medium. For glutamic acid, proline, glycine, alanine, and aspartic acid, which have particularly significant inhibitory effects, it was observed that lipase production was suppressed at concentrations of 500 ppm or more. Therefore, the concentration of amino acids that inhibit lipase production should be kept at 1000 ppm or less, and the concentrations of glutamic acid, proline, glycine, alanine, and aspartic acid should be kept below 1000 ppm.
It is desirable to feed an organic nitrogen source so that the concentration is 500 ppm or less. Furthermore, if the substrate to be fed contains a large amount of components other than amino acids or peptides, such as carbon sources as described below, the total amino acid concentration and the individual amino acid concentrations can be maintained within the above ranges by feeding. Therefore, amino acids and peptides are used as nitrogen sources for the metabolism of carbon sources, and do not work effectively for lipase production, making it impossible to improve lipase productivity. Therefore, it is essential that the substrate to be fed contains amino acids or amino acids and peptides as a main component in a content of 40% or more, more preferably 60% or more, and carbohydrates in the substrate that serve as carbon sources, Sugars, organic acids, etc. are 20
% or less, preferably 10% or less. Currently, there are no sensors that can detect the concentration of each amino acid in the culture medium online. Therefore, a specific method for controlling the amino acid level as described above is to feed an organic nitrogen source at a constant flow rate, and after a certain period of culture, achieve the amino acid level as described above and increase the productivity of lipase. The constant flow feeding method is used to increase the concentration of bacteria, and the relationship between the bacterial cell concentration, the feeding amount, and the amino acid concentration in the medium is determined in advance, and the feeding amount is adjusted so that the amino acid level can be maintained at the specified amino acid level in accordance with the bacterial growth curve. There is a program fed-batch method that changes . Furthermore, since the organic nitrogen source applicable to the present invention contains amino acids or amino acids and peptides as main components as described above, ammonium ions are generated in the medium in response to the assimilation of these. Therefore, it is also possible to control the amino acid level in the medium by measuring the ammonium ion concentration or generation rate using an ammonium ion electrode and controlling the feeding amount based on the feedback. Microorganisms that produce the lipase used in the present invention include microorganisms of the genus Rhizopus, Aspergillus, and Mucor. Furthermore, the lipase whose productivity has been increased according to the present invention showed a particularly remarkable increase in activity for non-aqueous reactions such as transesterification of oils and fats. [Examples] Next, the present invention will be explained in more detail using Examples, but the present invention is not limited to these Examples. Example 1 Using meat extract (manufactured by Wako Pure Chemical Industries, Ltd., amino acid content 45%) as an organic nitrogen source, the culture medium composition shown in Table 1, pH 5.6, temperature 30°C, stirring number 400 rpm, aeration amount
Rhizopus chinensis (Rhizopus
chinensis) IFO4768 was cultured in batch and constant flow fed-batch cultures. In the fed-batch culture method, Rhizopus chinensis from the pre-culture was inoculated into medium 3 containing only inorganic salts and oleic acid, and 15 c.c./fed-batch organic nitrogen source with a meat extract concentration of 120 g/mt was added. The cells were cultured in a fed-batch manner for 67 hours at a speed of 100 hrs., and an organic nitrogen source was supplied in approximately the same amount as in batch culture. After starting culture
Bacterial cells in the culture solution were sampled every 10 hours.
It was separated by suction filtration. The obtained bacterial cells were washed twice with tap water and then twice with an 80% acetone aqueous solution, and then vacuum-dried at room temperature for 24 hours. Using the dried bacterial cells thus obtained, a transesterification reaction was carried out in the reaction system shown in Table 2. A comparison of the transesterification reaction rates obtained is shown in FIG. As can be seen from FIG. 1, the rate of transesterification increased rapidly with constant flow fed-batch culture.

【table】

[Table] Example 2 The organic nitrogen source of Example 1 was yeast extract (Daigo Nutrition).
Batch culture and fed-batch culture of Aspergillus niger IFO4343 were performed using the same medium composition and culture conditions as in Example 1, except for using the same medium composition and culture conditions as in Example 1. In fed-batch culture, instead of constant flow feeding as in Example 1, a program generator is used to perform program control in which the fed-batch amount is varied according to a curve as shown in Figure 2. Ta. Other fed-batch culture procedures were the same as in Example 1. FIG. 3 shows a comparison of the transesterification ability of dried bacterial cells prepared in the same manner as in Example 1 in the case of batch culture and in the case of program-controlled fed-batch culture.
A high rate of transesterification was obtained by program control from the early stage of culture. Example 3 Proextract P (Banshu Seasoning Co., Ltd.) as an organic nitrogen source
Batch culture and fed-batch culture of Mucor javanicus IFO4569 were carried out using a commercially available commercially available product, Amino acid content 63%). Batch culture was carried out in the same manner as in Example 1, and fed-batch culture was carried out using an ammonium ion electrode to adjust the ammonium ion concentration in the medium to 1000 ppm.
An organic nitrogen source was fed under -off control.
The transesterification ability of dried bacterial cells prepared in the same manner as in Example 1 is shown in FIG. 4 for comparison. Lipase productivity could be increased by feeding ammonium ion concentration as an indicator.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between culture time and transesterification reaction rate in batch culture and constant flow fed-batch culture in Example 1. Graph showing the relationship between adjusted feeding amount and culture time, 3rd
The figure is a graph showing the relationship between culture time and transesterification reaction rate in the case of batch culture and program-controlled fed-batch culture in Example 2.
Graphs showing the relationship between culture time and transesterification reaction rate in the case of batch culture and fed-batch culture are shown respectively.

Claims (1)

[Claims] 1. When culturing a microorganism that produces lipase,
To increase the lipase production ability of a lipase-producing microorganism by culturing while maintaining the total amino acid concentration in the culture solution at 5000 ppm or less by feeding an organic nitrogen source mainly composed of amino acids or amino acids and peptides. A method for producing lipase characterized by: 2 The microorganisms that produce lipase are Rhizopus and Aspergillus.
or a microorganism belonging to the genus Mucor.